猪囊虫酸性核糖体磷蛋白基因P2重组杆状病毒的构建及鉴定

Construction and identification of recombinant baculovirus of acidic ribosomal phosphoprotein P2 from Cysticercus cellulosae

用Oligo(dT)软件设计了 1对酸性核糖体磷蛋白P2 (arpP2 )基因特异引物 ,以pUC18 P2为模板 ,经PCR扩增出 36 6bp的猪囊虫arpP2片段 ,酶切回收后与经过相同处理的 pFASTBAC 供体质粒连接 ,转化DH5α感受态细胞 ;对重组质粒经酶切和PCR鉴定后再转化DH10BAC 感受态细胞 ,提取高分子BacmidDNA ,用Lipofectin法转染Sf9单层昆虫细胞 ,待出现细胞病变后收集培养上清液 ,重新感染昆虫细胞 ,提取细胞DNA进行PCR鉴定 。

P2 gene was amplified with P2 specific primer by polymerase chain reaction (PCR) and was inserted into baculovirus expression vector pFASTBAC. Then recombinant plasmid was identified by digesting and PCR. The plasmid pFASTBAC P2 was transformed into DH10BAC competent E.coli cells. High molecular weight plasmid DNA was extracted from overnight cultures from E.coli white colonies ,which was named Bacmid P2 .The Bacmid P2 DNA was transfected intoSpodoptera fragiperda(Sf 9) cell to harvest virus supernatant and infect fresh Sf 9 cell again.The infected cell DNA was prepared and PCR was performed to identified recombinant virus. The result showed that recombinant virus with P2 was constructed successfully.